Trypanosomatid parasites cause some of the most challenging neglected diseases worldwide. The human- pathogenic triumvirate of Trypanosoma brucei spp., Trypanosome cruzi, and Leishmania spp. affect hundreds of thousands of individuals, causing human African trypanosomiasis, Chagas? disease, and a range of visceral to cutaneous lesions, respectively. With no approved vaccines and treatments challenged by resistance, delivery, and efficacy concerns, discovery of new drugs is critical for therapy, control, and elimination of these diseases. New, validated targets are needed to enter the discovery pipeline. We have recently shown that the protein kinase, AEK1, which is both highly conserved in these agents yet different from human protein kinases, is essential in the pathogenic bloodstream stage of T. brucei in vitro. We have also chemically validated it as important during infection. With this evidence, it is now important to understand the mechanisms of regulation of AEK1 and the pathways in which it functions. Although multiple protein kinase inhibitors are FDA-approved, the perils of off-target effects on other kinases remain a concern. Therefore we propose to investigate a proposed allosteric site on AEK1 by functional and biochemical studies. Such a site might offer a more specific means to block the AEK1 pathway. The AEK1 pathway is important in the T. brucei cell cycle. Our data shows that inhibition of AEK1 leads to inhibition of one or more events required for cytokinesis. To place its action within the cellular context, we will pinpoint the cytologic defect that occurs when AEK1 is inhibited. Because protein kinases exert their functions via modulating downstream effector molecules, the essentiality of AEK1 must reflect essentiality of one or more of its substrates. To identify substrates, we will use a genetically modified AEK1 that specifically accepts large thio-ATP analogs to enable specific thiophosphorylation of its substrates. Substrates will then be captured by virtue of the transferred thiophosphate. Identification of the thiophosphorylated peptides by mass spectrometry will allow development of a list of candidate substrates and a consensus phosphorylation site sequence that will enable identification of additional candidates. The identified molecules will be reviewed for their relationship to the phenotypic processes identified, as well as druggability, to aid in prioritization for future studies. By revealing mechanisms of regulation and identifying candidate substrates, this project will inform us about the pathway in which AEK1 functions. Given the high similarity of AEK1 across trypanosomatids, it is likely that many of our findings will be applicable to those pathogens.